PDBsum entry 1m0e

Transferase/DNA

PDB id: 1m0e

Contents

Protein chain

327 a.a. *

DNA/RNA

Ligands

SAH

Waters ×245

* Residue conservation analysis

References listed in PDB file

Key reference

Title

Zebularine: a novel DNA methylation inhibitor that forms a covalent complex with DNA methyltransferases.

Authors

L.Zhou, X.Cheng, B.A.Connolly, M.J.Dickman, P.J.Hurd, D.P.Hornby.

Ref.

J Mol Biol, 2002, 321, 591-599. [DOI no: 10.1016/S0022-2836(02)00676-9]

PubMed id

12206775

Abstract

Mechanism-based inhibitors of enzymes, which mimic reactive intermediates in the reaction pathway, have been deployed extensively in the analysis of metabolic pathways and as candidate drugs. The inhibition of cytosine-[C5]-specific DNA methyltransferases (C5 MTases) by oligodeoxynucleotides containing 5-azadeoxycytidine (AzadC) and 5-fluorodeoxycytidine (FdC) provides a well-documented example of mechanism-based inhibition of enzymes central to nucleic acid metabolism. Here, we describe the interaction between the C5 MTase from Haemophilus haemolyticus (M.HhaI) and an oligodeoxynucleotide duplex containing 2-H pyrimidinone, an analogue often referred to as zebularine and known to give rise to high-affinity complexes with MTases. X-ray crystallography has demonstrated the formation of a covalent bond between M.HhaI and the 2-H pyrimidinone-containing oligodeoxynucleotide. This observation enables a comparison between the mechanisms of action of 2-H pyrimidinone with other mechanism-based inhibitors such as FdC. This novel complex provides a molecular explanation for the mechanism of action of the anti-cancer drug zebularine.

Figure 1.
Figure 1. The reaction pathway of C5 MTases in the presence and in the absence of mechanism-based inhibitors. (a) The reaction pathway for all C5 MTases involves the transfer of the labile methyl group from S-adenosyl- Image -methionine (AdoMet) to the 5 position of the cytosine ring, proceeds through a covalent intermediate at position C6.[14] The nucleophilic attack upon the C6 position of cytosine drives the subsequent acquisition of the labile methyl group from AdoMet. (Note, the protonation status of Glu119 in M.HhaI[36]). (b) The inhibition by FdC. Following covalent complex formation and methyl transfer, the analogue remains bound to the active-site Cys, since abstraction of F cannot be achieved. (c) The inhibition by AzaC. Following covalent complex formation at a C6 with enhanced reactivity, slow methyl transfer may take place, but there is no H at C5 to abstract and the covalent complex persists. (d) The inhibition by zebularine. Following covalent complex formation at a C6 with enhanced reactivity as with AzaC, facilitated deamination at C4 cannot proceed, [33] since the amino moiety is absent from the analogue. Note that the water molecule nearest to the C4 atom is 3.6 Å away and the water molecule nearest to the C5 atom is 3.3 Å away.

Figure 2.
Figure 2. Structure of M.HhaI-AdoHcy-DNA containing zebularine. (a) The structural impasse between the proposed mechanism (outlined in Figure 1) and the barrier to substrate access in duplex DNA was overcome elegantly by the phenomenon of protein-induced base flipping. [15] Once the target base (zebularine here) is released from the constraints of the Watson-Crick base-pair, conventional active-site chemistry is facilitated. (b) Zebularine difference electron density maps (F[o] -F[c], a[c]) superimposed on the refined coordinates with carbon atoms being yellow, oxygen atoms red, nitrogen atom blue, and sulfur atom green, respectively. The blue electron density map contoured at 5.0s was computed with the zebularine moiety omitted from the atomic model. The green electron density maps contoured above 5.5s were calculated with the C4, C5, and C6 atoms of zebularine omitted from the atomic model, respectively. The zebularine is constrained in the plane of the ring by a highly conserved network of hydrogen bonds (via E119 and R165) and van der Waals interactions between the main-chain C=O group of F79 and C4 and C5 atoms. (c) A view perpendicular to (b), looking edge-on at the flipped zebularine molecule. A covalent bond is observed between C6 of the zebularine ring and an invariant thiolate side-chain C81, approaching the C6 perpendicular to the ring. The red electron density map contoured above 10.0s was calculated with the sulfur atom of C81 omitted from the atomic model.

The above figures are reprinted by permission from Elsevier: J Mol Biol (2002, 321, 591-599) copyright 2002.